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The thymus is a primary or central lymphoid organ in which T lymphocytes undergo diffentiation and maturation autonomously within the cortex, without the need for antigenic stimulation, and it is essential for the normal development and function of the immune system. The thymus has been shown to be a sensitive target organ following exposure to immunotoxicants and endogenous corticosteroids, and a decrease in size or weight is often one of the first noted measures of compound-induced effects with cortical lymphocytes (thymocytes) being especially susceptible. Therefore, changes in thymus histopathology and architecture are considered to be of particular relevance for immunotoxicity screening. The separate compartments in each lymphoid organ should be evaluated separately and descriptive rather than interpretive terminology should be used to characterize changes within those compartments (Haley et al., 2005). Therefore, enhanced histopathological evaluation of the thymus involves the determination of the size and cellularity of the cortex and medulla, which should be noted separately. Other changes to evaluate include, but are not limited to, increased lymphocyte apoptosis, lymphocyte necrosis, cortex:medulla ratio and an increase or decrease in the epithelial component of the thymus.
The thymus has been shown to be a sensitive target organ following exposure to immunotoxicants and a decrease in size or weight is often one of the first noted measures of toxicity (Schuurman et al., 1992). Moreover, lymphocytes (thymocytes) within the thymic cortex appear to be especially susceptible to the action of toxic compounds, both directly and indirectly (via the release of endogenous corticosteroids). Therefore, changes in thymus histopathology and architecture are considered to be of particular relevance for the determination of immunotoxicity (Van Loveren et al., 1996; Vos et al., 1997; Harleman, 2000, Kuper et al., 2000). Recent studies that examined the sensitivity of enhanced histopathology in the immune system of B6C3F1 mice demonstrated that the most consistent and discernable lesions were noted in the cortex of the thymus (Germolec et al., 2004).
It has also been shown that the histological findings in the thymus associated with a variety of different pharmaceutical agents correlate well with thymus weight and peripheral lymphocyte counts in both the rat and dog (Wachsmuth, 1983). However, since the thymus is an organ that is sensitive to the effects of stress (endogenous corticosteroids) and aging, it is very important to differentiate chemical-induced thymic atrophy from stress-related lymphocyte apoptosis and age-related thymic involution. Due to the potential difficulties of differentiating age-related changes from chemical-related effects, it may be best to conduct enhanced histopathology of the immune system in shorter-term studies, such as 14-day, 28-day or 3-month bioassays. As with all histopathological evaluations, comparison with control tissues is crucial.
According to the STP position paper: Best Practice Guideline for the Routine Pathology Evaluation of the Immune System (Haley et al., 2005), the separate compartments in each lymphoid organ should be evaluated separately and descriptive rather than interpretive terminology should be used to characterize changes within those compartments. Therefore, enhanced histopathological evaluation of the thymus involves the determination of the size and cellularity of the cortex and medulla, which should be noted separately. Refer to Pearse for more detailed information on the normal structure and function of the thymus (Pearse, 2006).
Decreased cellularity of the thymus is the most frequently encountered histologic finding associated with compound-induced effects on the thymus. Because decreased cellularity is often associated with the histologic presence of dead lymphocytes it is important to attempt to distinguish between lymphocyte apoptosis versus necrosis. The presence, severity grade and location of cell death, when present, should be determined. The determination of the type of cell death is important because it may provide insight into the pathogenesis of the lesion. The STP Committee on the Nomenclature of Cell Death recommends the use of the term “necrosis” to describe findings comprising dead cells in histological sections, regardless of the pathway by which the cells died (Levin et al., 1999). They also recommend the use of the modifiers “apoptotic” and “oncotic” to specify the predominant morphological cell death pathway.
Oncotic necrosis is the cellular process that can be seen in areas of thymus infarction or as a direct treatment-related effect and may or may not be accompanied by an inflammatory response rich in neutrophils. With oncotic necrosis, there is cell swelling and rupture of the cell membrane and subsequent release of cytoplasmic contents into the surrounding interstitium which incites the inflammatory response. Apoptotic necrosis on the other hand, is characterized by cell shrinkage, nuclear fragmentation, extrusion of membrane-bound cytoplasm and nuclear debris in the form of small dense apoptotic bodies. This process is typically accompanied by tingible body macrophages (defined as macrophages containing stainable cellular debris), which give the tissue a “starry sky” appearance (Figure 1c). It is therefore recommended that, whenever possible, a diagnosis of lymphocyte oncotic necrosis be reserved for those cases where treatment results in the classical form of necrosis rather than apoptosis.
As noted before, decreased numbers of lymphocytes in the thymus, leading to decreased cell density, decreased cellularity, or decreased compartment size, may be the result of direct thymic lymphocyte toxicity or may result from endogenous glucocorticoid release or age-associated involution. Certain chemicals that lead to direct thymus lymphocyte toxicity may result in increased numbers of apoptotic lymphocytes and tingible body macrophages (Figures (Figures11 and and2)2) or may result in oncotic necrosis (Figure 3). While not an absolute truth, apoptotic necrosis is most likely to occur as a secondary response to stress, while that of oncotic necrosis may be considered to be more representative of direct lymphocyte (thymocyte) toxicity. However, apoptotic necrosis and oncotic necrosis are not mutually exclusive processes and thus may occur simultaneously since both represent morphologic expressions of a shared biochemical network (Zeiss, 2003).
Endogenous glucocorticoid release in response to stress and debilitation can occur within a group of animals and this can result in increased numbers of thymus cortical apoptotic lymphocytes. However, lymphocytes in the cortex normally undergo numerous cell divisions before entering the medulla and apoptosis is a normal but usually minimal finding in this population of rapidly dividing cells. Therefore, an increase in the number of apoptotic cells should be noted only after comparison with controls. Although various methodologies are available for evaluating apoptosis (stained resin sections, DNA laddering, TUNEL, annexin V, caspase-3 activity as-says, mitochondrial assays, vital dyes and lysotracker red), each assay has its advantages and disadvantages that can render it appropriate and useful for one application but inappropriate or difficult to use in another (Watanabe et al., 2002). Transmission electron microscopy (Figure 2) is considered the “gold standard” for the evaluation of apoptosis.
Spontaneous aging and end-stage or chronic experimentally induced non-neoplastic thymic lesions are often morphologically similar with reduction in thymic weight and histological depletion of cortical lymphocytes. Therefore comparison with age-matched controls is crucial. The mechanisms responsible for age-related thymic involution are not known however sex hormones are involved. Orchidectomy in rats will cause involutional effects on the accessory sex organs with trophic effects on the thymus due to removal of the sex hormones. Treatment of old male rats with a stable analogue of luteinizing hormone-releasing hormone (LHRH) and the subsequent decrease in testosterone has been shown to result in regeneration of the thymus (Greenstein et al., 1987). Estrogen also has significant immunomodulatory properties, including induction of thymic involution (Yao and Hou, 2004).
An increase or decrease in the cortex:medulla ratio is another parameter that can be determined and recorded. However, within each lobule, the plane of section results in variation of this ratio when measured at multiple points. Therefore, in order to obtain an accurate ratio, a qualitative assessment in which the average of multiple ratios is determined or a quantitative morphometric analysis of the cortical and medullary areas would have to be determined as described by Pulido et al. (2005). To qualitatively evaluate this ratio, the functional lobule with an outer cortex and inner medulla should be examined. In general, the medulla normally occupies about one-third of the lobular volume in a typical adult rodent. A normal cortex:medulla ratio would therefore be close to 2:1. However, due to tangential cuts that can occur within any given lobule, all lobules should be examined and an overall ratio determined (Figure 4). In general, moderate to severe changes can be easily detected and graded qualitatively but the minimal-to-mild changes can be difficult to assess but can be detected by quantitative morphometric measurements. As always, a careful comparison with controls is needed to accurately identify a no effect level for the change.
An increase in the numbers of lymphocytes can occur within the cortex or medulla and can be focal or diffuse (Figures (Figures55--7).7). These lesions should be distinguished from lymphoma and the focal lymphocyte hyperplasia that accompanies age-related thymic atrophy. An increase in lymphocyte numbers in the thymus is usually in response to antigenic stimulation, accompanying inflammation, tumors, etc., and the cell population is mixed in contrast to the more homogeneous neoplastic population.
An increase in the numbers of epithelial cords and tubules within the medulla is another feature to evaluate (Figure 8). The loss of medullary lymphocytes can result in the epithelial component of the medulla appearing more prominent, but not necessarily with an increase in the number or size of epithelial cells. Since prominent and hyperplastic medullary epithelial cells are also a common age-related change found in association with thymus involution, consideration of age and comparison with controls should help to determine if this histological change is test article or age-related. The proliferation of thymic epithelial cells should also be differentiated from the neoplastic epithelial component of thymomas in which the cells can be spindle-shaped, ovoid or polygonal in shape with large nuclei or undergoing squamous differentiation.
The epithelium-free areas (EFA) in the thymus are lymphocyte-rich regions, devoid of stromal elements, non-vascularized, and with unknown function (Figure 9; Bruijntjes et al., 1993). It is postulated that they may be lymphocyte reservoirs (Van Ewijk, 1984), proliferation sites of lymphocytes (Duijvestijn et al., 1982; Godfrey et al., 1990) or a specific intrathymic pathway for T lymphocytes (Bruijntjes et al., 1993). These EFAs are located in the subcapsular region and serial sections show that they run from the subcapsular area to deep in the cortex, often bordering the medulla (Bruijntjes et al., 1993). The occurrence and extent of EFAs varies between rat strains. They have been identified in the Wistar rat, diabetes-prone and diabetes-resistant rats however they have not been identified in the WAG/Rij rats. The occurance and extent of EFAs can also vary with age. In Wistar rats EFAs are extensive whereas in rats over 17 months of age these areas have not been found. Some EFAs may be evaluated during the initial H&E screen however many EFAs may be overlooked using this method alone (F. Kuper, personal communication). Therefore, for an in-depth examination to allow for the accurate identification and evaluation of this compartment, special stains would be needed. Keratin and laminin stains for stromal elements would be negative and a stain for MHC class II cells would be negative except for a few cells (Bruijntjes et al., 1993).
The presence, severity grade and location of other cells and lesions, such as pigment, extramedullary hematopoiesis, cysts, etc. should also be noted. An example of a checklist for the changes to be noted in the thymus for enhanced histopathology is given in Table 1. This table is intended to be an example of a guideline, but not a rigid checklist, that the pathologist can use during histological evaluation rather than a format for reporting lesions. The diagnoses listed in this table are descriptive rather than interpretive, consistent with the STP position paper: Best Practice Guideline for the Routine Pathology Evaluation of the Immune System (Haley et al., 2005).
This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.